Lysosomes are specialized intracellular organelles where proteins, various lipids (including glycolipids and cholesterol) and carbohydrates are degraded and recycled to their primary constituents that enable synthesis of new proteins, membrane components and other molecules. Lysosomes are also utilized by cells to help maintain homeostasis and cellular health through an adaptive cellular process known as autophagy that increases lysosomal activity to provide additional amino acids for increased biosynthesis of various proteins (e.g., antibodies and interferons) and to supply nutrients for energy production to deal with stressful periods of nutrient deprivation or viral infections. Each metabolic process is catalyzed by a specific resident lysosomal enzyme. Genetic mutations can cause deficiencies in lysosomal biological activities that alter metabolic processes and lead to clinical diseases. Lysosomal storage disorders (LSDs) are a class of approximately 50 different human metabolic diseases caused by a deficiency for specific lysosomal proteins that results in the accumulation of various substances within the endosomal/lysosomal compartments. Many of these diseases have been well-characterized to understand the deficient lysosomal protein and the resultant metabolic defect. For example, there are several LSDs of altered glycolipid catabolism such as Gaucher, Fabry, and Tay-Sachs/Sandhoff. Neimann-Pick C is characterized by impaired lipid and cholesterol metabolism while diseases of altered carbohydrate metabolism such as glycogen storage diseases type II (Pompe) and type III (Corey-Forbes) have also been characterized. Other LSDs alter metabolism of bone or extracellular matrices [e.g., mucopolysaccharidoses (MPS I-VII), Gaucher] and protein turnover (neuronal ceroid lipofuscinoses; Batten, etc.). While LSDs are relatively rare, they can cause severe chronic illness and often death if not effectively treated.
There are no known cures for lysosomal storage diseases but a number of different treatment approaches have been investigated for various LSDs including bone marrow and umbilical cord blood transplantation, enzyme replacement therapy (ERT), substrate reduction therapy (SRT) and pharmacological chaperone therapy. Gene therapy is also being developed but has not been tested clinically. Of these treatment approaches, ERT is the most established with multiple ERTs approved for the treatment of various LSDs including Gaucher, Fabry, Pompe, MPS I, MPS II and MPS VI while one SRT drug is approved for the treatment of Gaucher disease.
The concept of ERT for the treatment of a lysosomal storage disease is fairly straightforward where a recombinant human lysosomal enzyme is administered in patients to supplement the deficient biological activity and improve clinical symptoms. However, unlike other protein therapeutic treatments that function primarily at the cell surface or outside of cells (e.g., anti-VEGF and other antibodies, erythropoietin, clotting factors, etc.), lysosomal enzymes must function inside cells, within lysosomes, and therefore use a mechanism for entering cells from the outside and subsequent delivery to these internal compartments. In mammals, the branched carbohydrate structures on the protein backbone on certain asparagine residues (N-linked oligosaccharides; N-glycans) for most soluble lysosomal enzymes are post-translationally modified to form a specialized carbohydrate structure called mannose 6-phosphate (M6P). M6P is the natural biological signal for identification and transport of newly synthesized lysosomal proteins from the Golgi apparatus to lysosomes via membrane-bound M6P receptors. A class of M6P receptors (cation-independent M6P receptor; CI-MPR) also cycles to the plasma membrane and is functionally active for binding and internalizing exogenous lysosomal proteins. The CI-MPR is believed to have evolved to recapture lysosomal proteins that escaped cells (via secretion out of cells) and thus, provide a targeting mechanism for internalizing exogenous lysosomal proteins and is the basis for enzyme replacement therapy for various LSDs.
Recombinant lysosomal enzyme replacement therapies have been shown to be generally safe but their effectiveness for reducing clinical symptoms varies widely. For example: Fabrazyme™ (recombinant acid α-galactosidase A; Genzyme Corp.) ERT dosed at 1 mg/kg body weight every other week is sufficient to clear accumulated substrate from endothelial cells in Fabry disease while 40 mg/kg of Myozyme™ (recombinant human acid α-glucosidase, rhGAA; Genzyme Corp.) dosed every other week is only moderately effective for Pompe disease. The disparate efficacy is primarily attributed to differences in the M6P content such that low levels of M6P correlates with poor drug targeting and lower efficacy. The manufacture of recombinant lysosomal enzymes is very challenging because it is extremely difficult to control carbohydrate processing, particularly the level of M6P in mammalian expression systems. Two specialized Golgi enzymes catalyze the M6P modification; N-acetylglucosamine phosphotransferase adds phosphate-linked N-acetylglucosamine onto certain terminal mannose residues while N-Acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (also known as Uncovering Enzyme) removes the covering N-acetylglucosamine to reveal the M6P signal. However, N-acetylglucosamine phosphotransferase is limiting in cells and this biochemical reaction is inherently inefficient for various lysosomal proteins. Over-expression of lysosomal proteins during the manufacturing process greatly exacerbates this problem and leads to highly variable amounts of M6P. Consequently, carbohydrate processing is typically incomplete and leads to the production of recombinant lysosomal enzymes with mixtures of N-glycans that contain M6P, non-M6P structures of high-mannose type N-glycans and complex-type N-glycans (typical for secretory proteins). To complicate matters, dead or damaged cells release enzymes such as phosphatases into the cell culture medium which remove M6P. Consequently, reduced M6P content lowers the binding affinity of a recombinant lysosomal enzyme for M6P receptors and decreases its cellular uptake and thereby, reduce drug efficacy. Dead or damaged cells release other glycosidases that remove other carbohydrates (e.g., sialic acids, galactose, etc.) to reveal internal carbohydrates that are not typically exposed and these N-glycans are readily identified as aberrant. These incomplete N-glycan structures increase the clearance rate of recombinant lysosomal proteins from the circulation which can also reduce drug efficacy. Higher drug doses are therefore necessary to compensate for reduced efficacy. Higher drug dose requirements however have multiple negative implications: (1) higher drug dose could be cost-prohibitive by increasing an already expensive treatment; (2) high drug doses require long infusion times; (3) large amounts of circulating drug results in significant antibody responses (seen in most Pompe patients) and numerous patients have also experienced allergic reactions during infusions. The FDA has issued a “black-label warning” for Myozyme and the drug is typically administered very slowly at the beginning but ramped up over the course of the infusion. This strategy helps to mitigate the allergic responses but significantly lengthens infusion times where 12-hr infusions are not uncommon.
One potential strategy for improving drug targeting for various lysosomal ERTs employs a targeting peptide to efficiently target ERTs to lysosomes without requiring the traditional M6P carbohydrate structures. This is conceptually feasible since the cation-independent M6P receptor contains a distinct binding domain for a small peptide called insulin-like growth factor 2 (IGF-2) and this receptor is therefore known as the IGF-2/(IGF-2/CI-MPR). This receptor is in fact solely responsible for internalizing exogenous M6P-bearing lysosomal proteins because the IGF-2/CI-MPR is present and biologically active on the cell surface. The other class of M6P receptors, the cation-dependent M6P receptor (CD-MPR), is only involved in the transport of lysosomal proteins within cells because it is not biologically active on cell surfaces and lacks the IGF-2 peptide binding domain. The IGF-2/CI-MPR has two separate binding sites for M6P (domains 1-3 and 7-9, respectively) such that it binds a mono-M6P N-glycan (1 M6P residue on N-glycan) with moderate affinity or a bis-M6P N-glycan (two M6P residues on the same N-glycan) with approximately 3000-fold higher affinity. Since lysosomal proteins contain mixtures of complex (no M6P), mono- and bis-M6P N-glycans, their affinities for the IGF-2/CI-MPR vary widely depending on the type and amount of M6P-bearing N-glycans.
There remains a need to develop strategies to create IGF-2-linked proteins for improved protein targeting. There remains a need for constructs that maintain the correct protein conformation.